Method for coating medical implants

ABSTRACT

The present invention relates to a method for coating a medical implant, wherein the implant is submersed in an aqueous solution of magnesium, calcium and phosphate ions through which a gaseous weak acid is passed, the solution is degassed, and the coating is allowed to precipitate onto the implant. The invention further relates to a medical implant coated in said method and to a device for use in said method.

FIELD OF THE INVENTION

[0001] The invention relates to a method for coating implant materialswith carbonated calcium phosphate films. More in particular, it isconcerned with the use of carbon dioxide gas—a weak acid—to decrease thepH of aqueous supersaturated calcifying solutions and deposit carbonatecontaining calcium phosphate layers onto implants during the naturalrelease of carbon dioxide gas at physiological temperature. Furthermore,the invention describes a new coating method for improvingbiocompatibility and bone-bonding properties of medical implants, suchas orthopedic and dental prostheses.

BACKGROUND OF THE INVENTION

[0002] Calcium phosphates are the principal constituent of hard tissueslike bone, cartilage, tooth enamel and dentine. Naturally occurring boneminerals are made of sub-micrometer, poorly-crystalline carbonatedcalcium phosphate crystals with hydroxyapatite structure. However,unlike the synthetic and ideal stoichiometric hydroxyapatite Ca₁₀(PO₄)₆(OH)₂ with atomic Ca/P ratio of 1.67, the composition and crystallinityof bone mineral is significantly different. Bone minerals consist mainlyof a complex mixture of calcium ions, phosphate ions, carbonate ions,and hydroxyl ions and may be represented by the following formulae:

Ca_(8.3)(PO₄)_(4.3)(HPO₄, CO₃)_(1.7)(OH, CO₃)_(0.3).xH2O

[0003] It has been demonstrated that calcium phosphate coatings on metalimplants allow a rapid bone apposition due to their osteoconductiveproperty, as compared with bare implants, e.g. cemented-less proximalhip stems. In vivo and in contact with body fluids, a thin layer ofbiological hydroxyl carbonated apatite is formed on the surface of someimplants, like bioactive glasses, hydroxyapatite ceramics. Subsequently,living bone tissue is directly apposite to this HCA layer. The directbone apposition onto and/or growth into the implant surface conduct tosome advantages such as a firm and immediate implant fixation and longterm result.

[0004] Several techniques, such as plasma spraying, flame spraying,electrophoretic deposition, magnetron sputtering and dipping, have beendeveloped for coating hydroxyapatite and others calcium phosphates ontoimplants. The most conventional coating method is plasma spraying.

[0005] A drawback of most hydroxyapatite-coated implants is that theanchoring of hydroxyapatite onto the implant requires elevatedprocessing temperatures, which limit the choice of substrate materialsand result in high processing costs. In the plasma-spraying process, theraw material i.e. hydroxyapatite, is once molten at a high temperatureso that the resulting apatite coatings are different in type from boneapatite. The coatings are frequently thick and brittle and are subjectedto fracture at the interface between coating and implant, e.g. betweenhydroxyapatite and titanium, thereby releasing large particles in thebody. Moreover, the method is rather unsuitable for numbers of polymersubstrates because of the high temperature involved. Furthermore, it isnot possible to incorporate biologically active agents, like proteins orantibiotics, within the coating, which may be useful to encourage bonein-growth or to prevent infection.

[0006] Additionally, most of these coatings are produced in a line ofsight process, thereby prohibiting uniform application of hydroxyapatiteon implants with complex surface geometry (e.g. porous surface). Theprevious methods have low efficiency for small and round-shapedsubstrates such as metallic dental implants.

[0007] The aim of the present invention is to provide a simple methodfor coating an implantable device with a thin, dense and bioactive layerof carbonated calcium phosphate. The said layers are processed atambient temperature by soaking the implantable devices into a calcifyingsolution where carbon dioxide gas is passed through. The producedbioactive coatings result in effective bone apposition and in-growth andthereby ensure bone-bonding properties to the implants. The implantabledevice can be used in a wide variety of biomedical applications(surgery, bone-replacement, prosthodontics, dental roots, crowns andorthopedic joints, etc).

[0008] Relevant Literature

[0009] The solubility products of the different calcium carbonatephosphate compounds are described as a function of pH, carbon dioxidepartial pressure and temperature in the publication of G. Vereecke andJ. Lemaitre, “Calculation of the solubility diagrams in the systemCa(OH)₂—H₃PO₄—KOH—HNO₃—CO₂—H₂O” J. Crystal Growth 104 (1990) 820-832 andin the contribution of F. C. M. Driessens entitled “Formation andstability of calcium phosphates in relation to phase composition of themineral of calcified tissue” in Calcium Phosphate Bioceramics, edited byK. de Groot, CRC Press (1984).

[0010] The publication of P. Serekian entitled “Hydroxyapatite coatingsin othopaedic surgery” edited by R. G. T. Geesink and M. T. Manley,Raven Press Ltd, New York (1993), p 81-97, discusses the advantages anddrawbacks of plasma and flame spraying, electrophoresis, dip coating andmagnetron sputtering.

[0011] EP No. 0 389 713 B1 (Kokubo, 1989) describes a process forapplying a bioactive hydroxyapatite film on implant substrates ofinorganic, metallic or organic material, by soaking an assemblycomprising a glass, mainly comprising CaO and SiO₂, facing a substrateat a predetermined distance apart, in an aqueous solution substantiallysaturated or supersaturated with constituent ions of hydroxyapatite. Inthe method according to the present invention, it is not necessary toprovide an assembly of glass facing the substrate to be coated.

[0012] EP No. 0 450 939 A2 and corresponding U.S. Pat. Nos. 5,164,187and 5,188,670 (Norian, 1990, 1991) describe a complicated process andapparatus for coating porous substrates with a hydroxyapatite film. Thismethod comprises combining a soluble calcium ion source and a solublephosphate ion source, wherein the molarity of the calcium ions is in therange of about 0.05-5 M, the molarity of the phosphate ions is in therange of about 0.01-1 M, at the temperature of 60-90°C. and pH of 5-8.5,under conditions leading to controlled nucleation and modulated growthof hydroxyapatite needle-like crystals. Basically, one solution isinjected into a circulating medium, resulting in the precipitation ofhydroxyapatite whiskers or single-crystals that reach and cover thesurface to be coated. This prior art method has two important drawbacks.First, hydroxyapatite crystals precipitate in the solution. On theopposite, in the method according to the invention, crystals nucleatedirectly on the implant surface leading to superior interfacialattachment. Second, the coating, as described in the above process, ismade by stacking hydroxypatite crystals through a fluid stream which isessentially a line of sight process and thereby giving shadows effectson complex shaped surfaces. In the present invention, the deposition ofcarbonated calcium phosphate layers is not dependent on the direction offluid flow.

[0013] International patent application WO A,93 07912 (Sherwood Medical,1993) describes a bioimplant obtained by soaking a base material to becoated in a saturated or supersaturated solution of hydroxyapatite. Thebase material has been previously provided with an organic polymercontaining sulfonic or carboxyl groups. In the method according to theinvention, it is not necessary to first provide the implant to be coatedwith such an organic coating.

[0014] International patent application WO 95 13101 (de Groot, 1993)teaches a method for coating an implant substrate with a bioactivematerial represented by the general formulaCa_(p)(PO₄)_(q)(CO₃)_(r)(OH)_(s) in which p>1 and q, r and s>0, and inwhich 2p=3q+2r+s. The said substrate is soaked in a solution in which atleast calcium ions, preferably carbonate ions, and if required,phosphate ions are present, after which the bioactive material isprecipitated from the solution on the substrate by either heating thesolution or the substrate. In the present invention, the temperature isfixed within the range 5-50°C. and there is no need to heat the solutionor the substrate to induce the precipitation of calcium phosphate.Moreover, the feasibility and bioactivity of such a coating has not beenexperimentally demonstrated in the International Patent Application WO95 13101.

[0015] EP No. 0 678 300 A1 (Kokubo, 1994) discloses a process forproducing a bone substitute material. In essence, a primary surfacelayer of a titanium oxide phase and amorphous phases of alkali titanatesare formed by soaking a base material made of titanium or its alloy inan alkali solution and heating the base material to temperature lowerthan the transition point e.g. 300-800°C. Subsequently, the alkali- andheat-treated base material is immersed in aqueous solution whichcontains calcium and phosphorus ions to a level of, at least the apatitesolubility, and thus producing a second layer comprising apatite on topof the said primary surface layer.

[0016] The patent applications EP 972011425/2, U.S. Pat. No. 8,855,835and Canada 2,205,107 (Isotis BV) describe a nanotechnology process forimplant surface treatment which can subsequently induce theprecipitation of calcium phosphate layers by soaking in a calcifyingsolution. The implantable devices have a surface roughness beforecoating with an average peak distance between 10 and 1000 nm to inducethe precipitation of calcium phosphate layers.

[0017] Japanese patent application 08040711 discloses a process forforming a hydroxy apatite coating, wherein calcium phosphate isdissolved in a solution containing sodium hydroxide, by applying highpressure carbon dioxide gas. The coating is deposited by discharge ofcarbon dioxide gas. In this known process, sodium hydroxide is presentin the calcifying solution, which significantly increases the pH. As aresult, a high pressure of carbon dioxide is needed in order to obtain alow enough pH to dissolve sufficient calcium phosphate.

SUMMARY OF THE INVENTION

[0018] The object of the present invention is to provide a simple methodfor coating the surface of medical implants with bioactive carbonatedcalcium phosphate layers. The said coatings are produced by soaking theimplantable devices into highly concentrated calcifying solutions at lowtemperature. The calcifying solutions are composed of calcium,phosphate, magnesium, carbonate and additionally sodium chloride saltsdissolved into water by bubbling carbon dioxide gas. During the naturalrelease of carbon dioxide gas or its exchange with air, the pH of thecalcifying solution is increased and the saturation is raised until thenucleation of carbonated calcium phosphate crystals on the surface ofimplantable devices. The said layer deposited and growth onto themedical implants. The process of bubbling/releasing CO₂ gas through orfrom calcifying solutions can be repeated until a sufficient thicknesshas been reached. The present invention has the following advantagesover conventional coating techniques: it is simple and cost-effectiveapproach, no expensive and intricate pieces of equipment are needed. Itis a low temperature process applicable to various substrates. Further,it has been found that materials can be deposited on a substrate in thepresent process, which was hitherto impossible. Octacalcium phosphatecoatings, for instance, cannot be prepared with conventional plasmaspraying techniques, due to the heat instability of the coatingmaterial. Such coatings also have been found not to grow in epitaxialfashion when employing other coating techniques.

[0019] As the coating is applied by using a fluid, complex shapedimplants (porous or beaded surfaces) can be uniformly covered with athin layer of carbonated calcium phosphate. The obtained layer is strongand wear resistant. The said layer is formed by using a biomimeticapproach (physiological fluids, temperature and pH) and thus, abone-like apatite layer having a high reactivity and adsorption propertyis deposited on the surface of medical implants. The biocompatibilty andbone-bonding properties of such coated devices have been demonstrated byimplantation in animal models.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020]FIG. 1. Bioreactor for producing the bioactive carbonated calciumphosphate coating FIG. 2. pH changes and temperature as a function ofsoaking time in the calcifying solution and in pure water FIG. 3. SEMphotomicrograph of the metal surface (Ti6Al4V) coated with the bioactivecarbonated calcium phosphate layers

[0021]FIG. 4. XRMA spectra of the carbonated calcium phosphate coatingon Ti6Al4V

[0022]FIG. 5. SEM photomicrograph of a porous tantalum implant coatedwith the carbonated calcium phosphate layers

[0023]FIG. 6. XRMA spectra of the bioactive carbonated calcium phosphatecoating on porous tantalum implant

[0024]FIG. 7. FT-IR spectra of the bioactive carbonated calciumphosphate layers recorded on Ti6Al4V implant

[0025]FIG. 8. TF-XRD pattern of the carbonated calcium phosphate onTi6Al4V implant

[0026]FIG. 9. SEM photomicrograph of a Ti6Al4V surface covered withcalcium phosphate layer deposited using a calcifying solution notcontaining magnesium ions

DESCRIPTION OF SPECIFIC EMBODIMENTS

[0027] The bioactive carbonated calcium phosphate layers may be appliedto any medical implant, inorganic, metallic or organic materials. Theimplant may be flat, dense or of a complex shape. It may have a porous,beaded or meshed ingrowth surface.

[0028] Metals, such as stainless steel, titanium, nickel, cobalt,chrome, niobium, molybdenum, zirconium, tantalum, and combinationsthereof, can be coated with the carbonated calcium phosphate layers fororthopaedic and dental applications. For example, devices used in totalhip arthroplasty such as porous or non-porous acetabular cups and theproximal region of hip stems may be coated with the bioactive carbonatedcalcium phosphate layers.

[0029] Ceramic materials, such as alumina and zirconia, glasses such asbioactive glasses made of CaO—SiO₂—P₂O₅, and calcium phosphates, such ashydroxyapatite and tricalcium phosphate, may be coated with thebioactive carbonated calcium phosphate layers.

[0030] The subject coatings can be applied to various polymers andplastics, more preferably biocompatible or bioresorbable ones likepolyactive™.

[0031] Before applying the coating, the substrates are preferablycleaned or treated to remove any surface contaminants and to promotegood adhesion of the coating. Various methods for cleaning may beemployed. The metallic implants may be rinsed with a degreaser, i.e.acetone, alkyl alcohols, etc. and then thoroughly rinsed with purewater.

[0032] In order to improve coating adhesion, various surface treatmentsmay be applied to metal implants. Mechanical surface treatments, such assand-blasting, scoring, polishing and grinding can increase surfaceroughness of the implants and improve the bonding strength between thecoatings and metal substrate. For similar purposes, chemical surfacetreatments may be also applied to metal substrates prior to coating.Among others chemical treatments available for metals, acid etchingswill be preferred by treating implantable devices with strong mineralacids, such as hydrofluoric, hydrochloric, sulfuric, nitric andperchloric acids. It may also useful to treat the metal devices withoxiding agents such as nitric acid, peroxyhalogen acids,hydroxyperoxides, or hydrogen peroxide to form a fresh metal oxidelayer. After the mechanical or chemical treatment, it is necessary torinse the implants with pure water under ultrasounds for removal ofsurface contaminants.

[0033] The method for coating medical implants with bioactive carbonatedcalcium phosphate layers consists of soaking medical implants intocalcifying solutions at low temperature. This simple method is based onthe finding that calcium phosphates are more soluble in mildly acidicmedium than at neutral and even basic pH. Thus, aqueous solutions ofcalcium and phosphate ions can be more concentrated at mildly acid thanat neutral pH. In other words, calcium phosphates precipitate at neutralor basic pH while they remain soluble at mildly acidic pH from asolution having the same concentrations of salts.

[0034] An increase of pH in the solution can induce the followingstages: under-saturation, super-saturation or the formation of ameta-stable state, nucleation and crystal growth. Calcium phosphatenuclei can form onto a substrate—heterogeneous nucleation—when asolution has reached the super-saturation limit or the meta-stablestate. At the super-saturation state, crystals can subsequently growfrom metastable fluids. At higher saturation, homogeneous nucleation orprecipitation in the solution is the predominant process. This inventionmakes use of pH changes to control the above stages and to induce thedeposition of carbonated calcium phosphate layers on the surface ofmedical implants.

[0035] The above object can be achieved by bubbling a gaseous weak acid,preferably carbon dioxide gas, into a calcifying solution in order todecrease pH and thereby to increase the solubility of calcium phosphatesalts. It is well known that natural sparkling water has a mildly acidicpH resulting from dissolved carbon dioxide gas. It is also an importantfeature that the pH of mineral water slowly increases to neutral orslightly basic pH during the natural release or exchange of dissolvedcarbon dioxide gas with air.

[0036] In a number of preferred embodiments, the bubbling of carbondioxide gas into the calcifying solution is required. Carbon dioxide gaswill dissolve in the calcifying solution and form hydrogen carbonateions in water (equation (1) and (2)). The said medical implants areplaced into an aqueous calcifying solution in which a gaseous weak acid,such as carbon dioxide gas, is passed through to produce a weakly acidicmedia. The initial pH of said calcifying solution is maintained in therange 3-7, preferably about 5.5 to 6.5 by bubbling CO₂ gas (FIG. 2). Thecarbon dioxide gas is introduced into the solution at a sufficientpressure to continuously generate bubbles. The pressure of CO₂ gas willbe in the range 0.1-10 bars, preferably within 0.5 to 1.5 bars, morepreferably about 1 bar.

[0037] In a preferred embodiment, the bioactive carbonated calciumphosphate coatings on medical implants are produced into an improvedbioreactor or fermentor system (FIG. 1). A modified bioreactor systemfor culturing cells or micro-organisms is used for coating medicalimplants with bioactive carbonated calcium phosphate layers. Thebioreactor should be made of borosilicate glass or stainless steelcoated with Teflon™ to avoid deposition or incrustation of carbonatedcalcium phosphate on the inner side walls. The volume of the bioractorcan range within 1 to 500 liters, more preferably from 1 to 150 litersdepending on the number of medical implants to be coated. The use of adouble-jacketed bioreactor vessel ensures a constant temperature in thecalcifying solution. The temperature is controlled by a thermocouplelinked to a thermo-circulator capable of cooling and heating at thedesired temperature. The implants like hip stems or acetabular cups andthe like are hold by special hooks fixed on the head-plate of thebioreactor. Several medical implants can be coated with the bioactivecarbonated calcium phosphate layers in the same batch. The pH ofcalcifying solution is measured with a sterilizable combined glasselectrode. The pH values are measured as a function of time. Thebioreactor is equipped with a magnetically coupled stirring system. Agas-inlet pipe and a porous sparger are provided for producing tiny CO₂gas bubbles into the calcifying solution and thus increases the gasexchange surface or aeration of the calcifying solution. Anelectro-valve or solenoid valve controls the flow of carbon dioxide gasintroduced into the bioreactor. The flow of carbon dioxide gas can beregulated as a function of time or pH. The bioreactor vessel should havean aperture to avoid increasing the internal pressure and to allow thenatural release of carbon dioxide gas out of the calcifying solution.The head-plate of bioreactor is equipped with an outlet condenser toprevent evaporation in the calcifying solution. All the criticalparameters, like pH, temperature, carbon dioxide flow, calcium,phosphate and carbonate concentrations can be measured, recorded andcontrolled by an automated system (controller) as a function of time.Prior to applying the coating, the bioreactor and implants can besterilized by autoclaving under water steam. The usual sterilizationprocedure consists of autoclaving the bioreactor, accessories andimplants in a steam at 121°C. for 30 minutes. All the accessoriesmounted to the head-plates are isolated with o-ring joints and filtersto maintain sterility during the coating process.

[0038] In the method according to the invention, the presence ofmagnesium, calcium and phosphate ions in the calcifying solution isessential. Particularly, the presence of magnesium has been found to beimportant for controlling the crystal growth of the coating duringdeposition from the calcifying solution. An optimum control of crystalgrowth leads to a uniform, strong and wear resistant coating.Particularly, the attachment of the coating to the substrate isbeneficially effected by the presence of magnesium ions in thecalcifying solution. A coating prepared according to the invention,preferably has crystals having a size in the submicrometer range. In apreferred embodiment, additional inhibitors of crystal growth, such ascarbonate ions, may be incorporated in the calcifying solutions. Ifrequired, counter ions, like sodium and chloride might also be presentto provide a constant ionic strength.

[0039] Preferably, the calcifying solution is prepared while the gaseousweak acid is bubbled through, in order to avoid precipitation. Theintroduction of the gas decreases the pH of the solution and allows thecomplete dissolution of the magnesium, calcium and phosphate, andpossible other salts. Preferably, the bubbling is started at least 5minutes before, and during, the addition of the salts. Thus, the pH islowered to approximately 3-8, more preferably to 5.5-6.

[0040] Of course it is also possible to start the bubbling with thegaseous weak acid after the addition of the desired amounts of the saltsto the solution. Once the bubbling is started, in accordance with thisembodiment, it is important to ensure that the salts dissolvecompletely.

[0041] The calcifying solution is preferably prepared with ultra purewater and pure grade chemicals. The calcifying solution is preferablyfilter sterilized through a 0.2 microns filter membrane, prior to beingpumped into the bioreactor. The molar calcium to phosphorus ratio in thecalcifying solution is generally within the range 1-3, more preferablybetween 1.5 to 2.5. The concentrations of the ions in the calcifyingsolution are chosen such, that in the absence of the gaseous weak acid,the solution is super-saturated or oversaturated. The molarity of thecalcium source will generally be in the range 0.5-50 mM, preferablyabout 2.5 to 25 mM. The phosphate source will generally be from about0.5 to 20 mM, more preferably about 1 to 10 mM. The concentration ofmagnesium in the calcifying solutions will usually be within the range0.1-20 mM, more preferably about 1.5 to 10 mM. The carbonateconcentration will range from 0 to 50 mM, more preferably 0 to 42 mM.The ionic strength will be within the range 0.10-2 M, more preferably inbetween 0.15 to 1.5 M. The calcifying solution is preferably stirred toapproximately 10-1000 rpm, more usually 50 to 200 rpm. The temperatureis maintained at about 5-80°C., preferably in the range of about 5-50°C.

[0042] The carbon dioxide has a limited solubility in aqueous solutions.In contact with air, a carbonated aqueous solution is free of CO₂ orcompletely degassed within few hours depending on the surface ofsolution in contact with air. In the open bioreactor described herein,the complete exchange of dissolved CO₂ gas with atmosphere takesapproximately 8 to 48 hours, more preferably between 12 to 24 hours. Thenatural release of CO₂ gas causes the pH of the remaining solution toincrease (FIG. 2). In others words, saturation in the calcifyingsolution can increase until the precipitation of the bioactive layers onthe surface of implantable materials occurs. Optionally, air can bebubbled through the solution to degas or aerate the solution andaccelerate the escape, release or exchange of the gaseous weak acid. Theinitial and final pH values as well as pH changes with time depend onthe amount of carbonate and phosphate salts added to the calcifyingsolution. The buffering capability can be adjusted to a desired pH valueby adding more or less of phosphate and carbonate salts. The pH can bemaintained within the desired range by introducing carbon dioxide gas.In essence, the flow of carbon dioxide can be adjusted by using anelectro or selenoid valve piloted by the controller. During the naturalrelease of CO₂ gas out of the calcifying solution, the pH will increaseto about 6-10, more preferably about 7.5 to 8.5 after soaking for 24hours. The carbonated calcium phosphate layer will precipitate on thesurface of implantable devices at a pH value of within about 6.5-7.5.The said precipitation on the surface of medical implants is related toa heterogeneous nucleation step. The carbonated calcium phosphatecrystals might subsequently precipitate into the calcifying solution bya crystal growth process. In the invention, the heterogeneous nucleationis favored by the energetic stabilization of nucleus on the substrate.The high density of nucleation ensures a uniform deposition ofcarbonated calcium phosphate crystals onto the surface of medicalimplants. The above process can be illustrated by the followingequations:

CO₂ (g)<−>CO₂ (aq)  (1)

CO₂ (g)+H₂O<−>HCO₃ ⁻+H⁺  (2)

10 Ca²⁺+6 PO₄ ³⁻+2 OH⁻→Ca₁₀ (PO₄)₆ (OH)₂↓  (3)

[0043] The process of bubbling carbon dioxide gas into the aqueouscalcifying solution and escape of the carbon dioxide gas from thesolution can be repeated to deposit a subsequent layer of carbonatedcalcium phosphate minerals on the implantable material. In a methodaccording to the invention, it may be essential to control the pH andthereby the nucleation stage by bubbling CO₂ gas for various time. Thebubbling time is usually comprised between a few seconds to minutes,preferably about 1 to 600 seconds. The introduction of carbon dioxidecauses a decrease of pH while the pH of calcifying solution has atendency to increase naturally without bubbling CO₂ gas. The increase ofpH may be due to the natural exchange of CO₂ gas with atmosphere and thebuffering capability of the calcification solution. By adjusting thetime and flow of CO₂ gas introduced into the calcifying solution, the pHcan oscillate around a value ranging from 6 to 9, more preferably the pHof the calcifying solution can be maintained between 6.5 to 7.5. This pHoscillation is correlated to the nucleation stage of carbonated calciumphosphate crystals on the surface of medical implants. A high density ofnucleation is thereby provided and carbonated calcium phosphate crystalscan nucleate and grow onto the surface of medical implants. Homogeneouslayers can uniformly deposit on the implant substrate. The totalthickness of layers will preferably be within the range 0.5-100 microns,more likely 0.5 to 50 microns. While the layers are thin, usually below5 microns, the coatings can diffract the natural light forming coloredfringes ranging from blue to red colors. This diffraction of light issimilar to the phenomenon that may be observed when a drop of oil ispresent on water. For higher thickness, the layers give a shiny gray orwhite coloration.

[0044] The thin carbonated calcium phosphate layers can induce theprecipitation of subsequent layers by immersion into a second calcifyingsolution. In other words, the thin carbonated calcium phosphate layerscan serve as seed crystals for subsequent layers. The second calcifyingsolution is preferably super-saturated with respect to hydroxyapatite.Under the super-saturation conditions, crystal growth may take place,and thick, crystalline and uniform calcium phosphate layers can beproduced onto the surface of a medical implant. The second calcifyingsolution should contain calcium and phosphate salts with only smallamounts of, or even without, inhibitors of crystal growth, likemagnesium or carbonate. As the second, or further layer will bedeposited on a calcium phosphate coating (the first layer), a goodattachment is more easily achieved.

[0045] The second calcifying solution can be prepared in the absence orpresence of a gaseous weak acid, such as carbon dioxide. Preferably, thesecond calcifying solution is buffered at a physiological pH, around7.4, with an appropriate buffer, like tris(amino-ethane) and dilutedwith hydrochloric acid. The concentration of calcium ions in the secondcalcifying solution may range from 0.5 to 10 mM, more preferably from0.5 to 5 mM. The concentration of phosphate may range from 0.5 to 6 mM,more preferably from 0.5 to 3 mM. Magnesium and carbonate ions arepreferably present in concentrations below 1 and 5 mM, respectively.More specifically, magnesium might be present in an amount between 0.1and 3 mM, more preferably between 0.5 and 1.5 mM. Sodium chloride, orany suitable salt may be added to maintain the ionic strength of thesecond calcifying solution at a value of 0.05 to 0.5 mM, preferably 0.1to 0.2 mM.

[0046] The composition and crystal size of the layers will be stronglydependent on the amount of crystal growth inhibitors in the calcifyingsolutions.

[0047] In a preferred embodiment, the layers will be composed ofhydroxyl carbonate apatite with a poor crystallinity or amorphouscalcium phosphates containing magnesium and carbonate ions. Depending onthe ion concentrations and pH of the calcifying solution, a coating of acompound having the general formula (I) can be obtained:

(Ca)_(p) (Mg)_(q) (Na)_(r) (PO₄)_(x) (CO₃)_(y) (OH)_(z)  (I)

with p>1 and x, y and z>0

and in which 2p+2q+r=3x+2y+z

[0048] A series of salts having the general formulae (I) can be coatedto medical devices. For example, if p=10, q=0, r=0, x=6, y=0 and z=2,the above formulae gives the structural formula of hydroxy apatiteCa₁₀(PO₄)₆(OH)₂ with a calcium to phosphorus ratio of 1.67. If 8<p<10and 4<x<6, series of calcium deficient apatite resembling to bonemineral are obtained. The coating may be also composed of octacalciumphosphate (OCP) with the formulae Ca₈H₂(PO₄)_(6.5)H₂O which is involvedin the early stage of biomimeralization of calcified tissues. If p=1 andy=1, the formula represents calcium carbonate CaCO₃ having calcite,vaterite, aragonite structure or a combination thereof.

[0049] The chemical composition of the coating can be variable but thelayers always contain magnesium, calcium and phosphate ions. If desiredcarbonate ions may be also incorporated within the coating, additionallythe film can include traces of sodium and chloride ions. The amount ofcalcium and phosphorus will average between 20 to 40 and 10 to 30 weightpercent, respectively. The magnesium and carbonate contents in thecoating will be within the range of 0.1-5 weight percent and 0-7 weightpercent, respectively. The metal to phosphorus ratio (M/P withM=Ca+Mg+Na) will be within the range 1.00 to 2.00, more preferably inbetween 1.30 to 1.80.

[0050] The present coatings may incorporate a wide variety ofbiologically active agents, such as peptides, growth factors, bonemorphogenetic proteins, combinations thereof, and the like. The growthfactors will be co-precipitated within the layers on the surface ofimplantable devices and may serve as drug delivery systems. The gradualrelease of growth factors around the coated article can stimulatedosteoblasts cells and enhance bone healing. Furthermore, antibioticslike tobramycin, vancomycin, and the like can be also precipitatedwithin the coatings to prevent infection post-surgically. Generally, thegrowth factors and antibiotics will be solubilised in the calcifyingsolutions at a concentration of 1 μg/ml to 1 mg/ml.

[0051] The coating process described herein can deposit a variety ofcalcium phosphate compounds containing carbonate and others ions on thesurface of an implantable device. The layers will be similar incomposition and crystallinity with bone and teeth minerals and havedesired bioresorbability, bone-bonding properties to improve thebiological fixation of medical devices to living calcified tissue.

[0052] It has further been found, that coatings on medical implants,prepared in a biomimetic approach, such as the present process, haveosteoinductive properties. A biomimetic approach concerns a processresulting in a calcium phosphate coating that, to a certain extent,mimics calcium phosphates resulting from biological mineralizationprocesses, such as in bone or sea shells. This means that a biomimeticprocess often takes place at ambient temperature and results in acalcium phospate that resembles one, or a combination of the numerousnaturally occurring calcium phosphate compositions. A biomimetic coatingmay be prepared employing a solution that is rich in at least calciumand phosphorous ions, either or not in physiologic concentrations, andoptionally in the presence of nucleation promoting agents, such asbioactive glass particles. Examples of biomimetic approaches include theprocess as described herein, but also those described by Kokubo (see EPNo. 0 389 317 A1 and NP No. 0 678 300 A1).

[0053] It has now been found that a biomimetic approach leads to aspecific reactivity (e.g. dissolution-reprecipitation of calciumphosphate or adsorption of endogenous biologically active agents, suchas BMP's), biological conversion after implantation, morphology, surface(micro)structure and/or implant porosity of the coating, which inducesformation of bone cells, such as osteoblasts, from progenitor cells evenwhen the implants are provided in vivo in non-bony tissues. It hasfurther been found, that the present process for providing a coating ona substrate leads to a particular morphology and crystal orientation,that increases the osteoinductive character of the biomimetic coating.Further, certain coatings having specific chemical compositions, such asOCP coatings, lead to even greater osteoinductive effects.

[0054] This invention is illustrated by the following examples butshould not be construed to be limited thereto. In the examples, thepercentages are expressed in weight unless specified otherwise.

EXAMPLE 1

[0055] Pieces of titanium alloy are cut from a sheet of commerciallyavailable Ti6Al4V foil or rods. Ti6Al4V plates of 10×10×2 mm andcylinders of 5 mm in diameter and 10 mm in length are used. Ti6Al4Vwires of 1 mm in diameter are also coated with the bioactive carbonatedcalcium phosphate layers. Prior to coating, the implants are sand- orgrit-blasted to increase their surface roughness. The implants areultrasonically cleaned for 15 min in acetone, then ethanol (70%) andfinally pure water. The Ti6Al4V plates are then etched for 30 min in anultrasonic cleaner with a concentrated acid mixture containing distilledwater, hydrochloric acid (HCl, 36%) and sulfuric acid (H₂SO₄, 96%) witha volume fraction of 2:1:1. A soft etching procedure can be alternatelyapplied by soaking the implants into a mixture made of 994 ml of purewater, 2 ml of hydrofluoric acid (HF, 40%) and 4 ml of nitric acid(HNO₃, 50%). The etched Ti6Al4V plates were thoroughly washed with purewater. After etching and rinsing, all samples are placed into a 3 litersinsulated bioreactor and sterilized with stem at 121°C. for 30 minutes.The calcifying solution is prepared by dissolving 40.00 g of NaCl(99.9%) 1.84 g of CaCl₂.2H₂O (99.9%) 1.52 g of MgCl₂.6H₂O (99.9%) 1.06 gof NaHCO₃ (99%) and 0.89 g of Na₂PO₄.2H₂O (99.9%) in 1000 ml of purewater. The calcifying solution is pumped through a 0.2 microns membranefilter into the bioreactor. Carbon dioxide gas is introduced into thesolution at a pressure of 0.5-1.5 bar generating CO₂ bubbles. The pH ofthe solution is measured with an electrode and continuously monitored.The solution is maintained at pH 5.5-6.5 by the introduction of CO₂ gas.The temperature is controlled to 37°C. by using a thermocouple and aheating device. The calcifying solution is continuously stirred at 100rpm. he flow of CO₂ gas is stopped and the pH starts to increase slowly.After soaking for 24 hours, the pH of calcifying solution is within therange 7.8-8.6. After coating, the samples are ultrasonically cleaned indemineralised water for 10 minutes and dried at 50°C. for several hours.The thickness of the bioactive layers is measured by using Eddy-Currentinstruments. The coating has a thickness averaging between 1 to 5microns. The tensile bonding strength of the layers onto the substrateaverage between 40 to 65 Mpa. The morphology and composition of coatingare evaluated by using SEM together with XRMA (FIGS. 3 and 4). Dense anduniform carbonated calcium phosphate layer are observed on the surfaceof implants. The layers are composed of micrometer sized globules orspherules containing Ca, O, P, and traces of Mg, Na and Cl (FIG. 3).FT-IR spectra and TF-XRD determine the crystallinity of the coatings.The FT-IR spectra (FIG. 7) show featureless and wide carbonate andphosphate bands typical of poorly crystallised hydroxyl cabonate apatitesimilar to bone mineral. The TF-XRD patterns (FIG. 8) indicate thediffraction lines of the Ti6Al4V substrate and halo or bump located ataround 30 degrees (2 theta) characteristic of amorphous calciumphosphate or poorly crystallised hydroxyl carbonate apatite phase. Forimplantation purposes, the coated devices are sterilized by steam at121°C. for 30 minutes.

EXAMPLE 2

[0056] Porous tantalum implants (Hedrocel™, Implex Corporation,Allendale, N.J.) of respectively 2.5 and 5 mm in diameter and 5 and 10mm in length are used. The implants are ultrasonically cleaned for 10minutes in acetone, ethanol (70%) and finally pure water. The implantsare then placed into meshed bags and hold into the bioreactor system.After autoclaving, the implants are soaked into a calcifying solution asdescribed in example 1. After coating, the coated devices areultrasonically rinsed with pure water and sterilized with an autoclave.The SEM observations and EDAX analyses confirm the uniform deposition ofa well-attached dense calcium phosphate layer on and into the poroustantalum implants (FIGS. 5 and 6).

COMPARATIVE EXAMPLE 3

[0057] Three Ti6Al4V plates were successively cleaned in acetone,ethanol, and demi water. Next, the plates were etched, using a mixtureof hydrochloric acid and sulfuric acid, and thoroughly rinsed with demiwater.

[0058] A calcifying solution was prepared by dissolving 40.0 g of NaCl,2.95 g of CaCl₂.2H₂O and 1.80 g of Na₂HPO₄.2H₂O in 1000 ml demi water,while bubbling carbon dioxide gas through the solution at a pressure of0.5-1.5 bar.

[0059] The Ti6Al4V plates were soaked at 37°C. for 24 hours in thecalcifying solution, and finally rinsed with demi water. A calciumphosphate layer was found to partially cover the plates. As can be seenfrom FIG. 9, the coating was not uniformly deposited on the surface ofthe substrates. It was found that the coating was not well attached tothe Ti6Al4V surface of the substrates, and could be easily removed orscraped off.

EXAMPLE 4

[0060] Porous tantalum cylinders were coated in a procedure analogous tothat of example 1. After soaking in the calcifying solution (which hadthe same composition as the calcifying solution in example 1), a thickand crystalline biomimetic layer covered the tantalum cylinders evenly.The layer was composed of octacalcium phosphate (OCP,Ca₈(HPO₄)₂(PO₄)₄.5H₂O) crystals aligned perpendicularly from the surfaceof the substrate, as determined by SEM on a cross-sectioned cylinder.

EXAMPLE 5

[0061] Animal Experiments

[0062] The protocol provides for the evaluation, safety andeffectiveness of the bioactive layers applied to different biocompatiblesubstrates such as Ti6Al4V, porous tantalum and polyactive implants invivo. The bone-conducting ability and bone in-growth of the bioactivelayers are evaluated by using several animal models. Conventionalhistology techniques are used to compared bare and coated implants. Theexperiments and substrates are not limited hereto.

[0063] Intra-femoral implantation in rats:

[0064] Four Ti6Al4V wires coated as described in example 1 or non-coatedwith the bioactive carbonated calcium phosphate are press-fit implantedin the femur of rats (Fisher, F344, adult male, 200-250 g) for 4 weeks.After sacrifice, the femoral bone and implants are retrieved, rinsedwith phosphate buffered solution, dehydrated and embedded in resin(PMMA). The bone and coated implants are stained with alizarin andcross-sectionned into histology slides. The surface of bone in contactwith the coating is observed by light microscopy and measured by imageanalyses. The bone conducting ability of the coating is compared to bareimplants.

[0065] Intra-cortical implantation in goats:

[0066] After sterilization, the coated, as described in example 2, ornon-coated porous tantalum implants (5 mm diameter and 10 mm long) areimplanted into the femoral bone of mature goats. The implants areinserted in both proximal and distal regions of the femurs. 6 coated orbare implants are used per goat. After implantation for 2, 4, 8 and 16weeks, the animals are sacrificed and the bones are retrieved. The bonesare then washed with phosphate buffer solution, dehydrated with seriesof ethanol and embedded in PMMA. The bones are cross-sectioned by usinga microtome sawing machine. The implants surrounded by bone are thencarbon sputtered for SEM observations. The total bone ingrowth withinthe porous tantalum implants is observed by back-scattering scanningelectron microscopy and measured by using image analyses techniques. Thetotal bone in-growth of coated porous tantalum implants is finallycompared to bare implants. The results of the study indicate a mean bonein-growth of 35% for porous tantalum alone and 80% for implants coatedwith the bioactive layers after 8 weeks in vivo. These results show atwo-fold increase in bone in-growth for implants coated with thebioactive layers.

[0067] Intra-muscular implantation in dogs:

[0068] After sterilization, implants coated with an OCP layer asdescribed in example 4, and non-coated porous tantalum cylinders (5 mmdiameter and 10 mm long) are implanted into the thigh muscles of maturedogs (body weight about 20 kilograms). From each implant type, eight (8)cylinders are implanted (one per dog) and the survival time is threemonths. Surgery is performed under general anaesthesia and sterileconditions. Briefly, a longitudinal skin incision is made in the leg andthe thigh muscle is exposed by blunt dissection. A longitudinal incisionis made in the muscle fascia after which an intramuscular pocket iscreated in which the implant is inserted. The incision is sutured with afine silk thread to keep the implant inside the muscle pouch, the skinis closed with a silk suture and the wound is cleaned with iodinetincture. After three months, the dogs are terminated with anintra-abdominal injection of pentobarbital and the implants arecollected with surrounding tissues and labelled as indicated before theywere implanted. All samples are subsequently fixed in 10% bufferedformalin at 4°C., dehydrated through a graded series of ethanol toethanol 100% and embedded in methyl methacrylate (MMA). Undecalcifiedsections are made on a modified innerlock diamond saw and examined bylight microscopy.

[0069] Histological analysis of the sections reveal that the uncoatedTantalum cylinders are surrounded and invaded with fibrous tissue, whilede novo bone formation is absent. In contrast, the OCP coated porousTantalum cylinders reveal abundant de novo formed bone that is in directcontact with the OCP coated implant surface. Almost the entire surfaceof the OCP coated porous cylinders is coated with a layer of bone. Theseunique results clearly indicate that a biomimetic coating, in thisexample composed of OCP crystals that are oriented perpendicular to theimplant surface, can induce bone formation in a non-bony environment.Coating composition, reactivity (e.g. dissolution-reprecipitation ofcalcium phosphate or adsorption of endogenous biologically activeagents, such as BMP's), biological conversion after implantation,morphology, surface microstructure and/or implant porosity can beresponsible for the osteoinductive property of the implant by inducingthe differentiation of progenitor cells into osteogenic cells.

What is claimed is:
 1. A method for coating an implant comprising thesteps of (a) contacting the implant with an aqueous solution ofmagnesium, calcium, and phosphate ions; (b) passing a gaseous weak acidthrough the aqueous solution; (c) degassing the aqueous solution; and(d) allowing the magnesium, calcium, and phosphate ions to precipitateonto the implant to form a coating.
 2. The method of claim 1 wherein thegaseous weak acid is carbon dioxide.
 3. The method of claim 1 whereinthe implant is formed from one or more of metal, organic material,polymer or ceramic.
 4. The method according to claim 1 wherein thecalcium and phosphate ions are present in the aqueous solution in amolar ratio of between about 1 to about
 3. 5. The method according toclaim 1 wherein the calcium and phosphate ions are present in theaqueous solution in a molar ratio of between about 1.5 to about 2.5. 6.The method according to claim 1 wherein the aqueous solution comprisesabout 0.5 to about 50 mM calcium ions and about 0.5 to about 20 mMphosphate ions.
 7. The method according to claim 1 wherein the aqueoussolution comprises about 2.5 to about 25 mM calcium ions and about 1.0to about 10 mM phosphate ions.
 8. The method according to claim 1wherein the aqueous solution comprises about 0.1 to about 20 mMmagnesium ions.
 9. The method according to claim 1 wherein the aqueoussolution comprises about 1.5 to about 10 mM magnesium ions.
 10. Themethod according to claim 1 wherein the aqueous solution comprises nocarbonate ions or less than about 50 mM carbonate ions.
 11. The methodaccording to claim 1 wherein the aqueous solution comprises no carbonateions or less than about 42 mM carbonate ions.
 12. The method accordingto claim 1 wherein the aqueous solution comprises an ionic strength inthe range of about 0.1 to about 2 M.
 13. The method according to claim 1wherein the aqueous solution comprises an ionic strength in the range ofabout 0.15 to about 1.5 M.
 14. The method according to claim 1 whereinthe gaseous weak acid is passed through the aqueous solution at apressure of about 0.1 to about 10 bar.
 15. The method according to claim1 wherein the gaseous weak acid is passed through the aqueous solutionat a pressure of about 0.5 to about 1.5 bar.
 16. The method according toclaim 1 wherein the aqueous solution has a temperature in the range ofbetween about 5°C. to about 80°C.
 17. The method according to claim 1wherein the aqueous solution has a temperature in the range of betweenabout 5°C. to about 50°C.
 18. The method according to claim 1 whereinthe implant is treated by a mechanical or chemical surface treatmentprior to contacting the implant with the aqueous solution.
 19. Themethod of claim 18 wherein the implant is treated by sand-blasting,scoring, polishing or grounding.
 20. The method of claim 18 wherein theimplant is treated by contacting with strong mineral acid or anoxidizing agent in a manner to etch the implant.
 21. The method of claim1 wherein the coating comprises magnesium ions, calcium ions andphosphate ions and one or more ions selected from the group consistingof hydroxide, carbonate, chloride, sodium and potassium.
 22. The methodof claim 1 wherein the coating comprises one or more of amorphouscarbonate calcium phosphate, hydroxyapatite, calcium deficient andhydroxyl carbonate apatite, oroctacalcium phosphate, dicalcium phosphatedihydrate or calcium carbonate.
 23. The method of claim 1 wherein thecoating has a thickness of about 0.5 to about 100 microns.
 24. Themethod of claim 1 wherein the coating has a thickness of about 0.5 toabout 50 microns.
 25. The method of claim 1 further comprising the stepof contacting a coated implant with a calcifying solution comprisingcalcium and phosphate ions, and allowing a precipitate layer of calciumand phosphate ions to form on the coated implant.
 26. A device forcoating an implant comprising (a) reactor vessel; (b) heating elementoperatively connected to the reactor vessel; (c) implant support; (d)stirrer disposed within the reactor vessel; (f) inlet and outletoperatively connected to the reactor vessel; and (g) controlled sourceof carbon dioxide operatively connected to the inlet.